The calcium-cation antiporter (CaCA) family of proteins is important for the control of intracellular calcium signaling. Maintenance of Ca2+ homeostasis depends on the functioning of specific transport systems that regulate Ca2+. One member of this family, the cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1), is the primary mechanism for Ca2+ efflux from myocytes. As such, NCX1 is intimately involved in cardiac muscle relaxation and in preventing cellular Ca2+ overload. NCX1 has also been implicated in the genesis of some cardiac arrhythmias and has increased function in heart failure. This study will generate mechanistic details of a medically relevant protein from the CaCA family of exchangers, NCX1. In addition, we will develop new methodology for the expression, purification, crystallization and phase determination of the CaCA family. This technology will be directly transferable to other families of membrane proteins to yield high-resolution structures. In particular, we aim to achieve a 3D structure of the intracellular regulatory loop of NCX1, the primary regulator of Ca2+ extrusion from mammalian cardiac myocytes, as well as the structure of a complete exchanger from a prokaryotic member of the CaCA family. All hypotheses from the structural analysis will be tested by functional assays. The knowledge from these structure/function studies may lead to rational drug design and to the resolution of remaining unknown aspects of the function of this critical regulator of cardiac and neural physiology. PUBLIC HEALTH RELEVANCE: The CaCA family of proteins are ubiquitous and vital components of Ca2+ signaling pathways utilized for maintaining Ca2+ homeostasis in a variety of cell types. The mammalian Na+-Ca2+ exchanger (NCX), the best- characterized member of the family, is intimately involved in cardiac muscle relaxation and in preventing cellular Ca2+ overload. Determining the structure and regulatory function of CaCA family members in general, and NCX1 in particular, will allow for rational design of novel pharmaceuticals that may be used in regulating cardiac contractility in health and disease related to dysfunctional Ca2+ transport.